Selective Determination of Volatile Sulfur Compounds in Wine by Gas

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J. Agric. Food Chem. 2010, 58, 9454–9462 DOI:10.1021/jf102008r

Selective Determination of Volatile Sulfur Compounds in Wine by Gas Chromatography with Sulfur Chemiluminescence Detection TRACEY E. SIEBERT,* MARK R. SOLOMON, ALAN P. POLLNITZ,† AND DAVID W. JEFFERY †

The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA 5064, Australia. Present address: Forensic Science South Australia, 21 Divett Place, Adelaide, SA 5000, Australia.

Volatile sulfur compounds can be formed at various stages during wine production and storage, and some may impart unpleasant “reduced” aromas to wine when present at sensorially significant concentrations. Quantitative data are necessary to understand factors that influence the formation of volatile sulfur compounds, but their analysis is not a trivial undertaking. A rapid and selective method for determining 10 volatile sulfur-containing aroma compounds in wine that have been linked to “offodors” has been developed. The method utilizes static headspace injection and cool-on-column gas chromatography coupled with sulfur chemiluminescence detection (GC-SCD). Validation demonstrated that the method is accurate, precise, robust, and sensitive, with limits of quantitation around 1 μg/L or better, which is below the aroma detection thresholds for the analytes. Importantly, the method does not form artifacts, such as disulfides, during sample preparation or analysis. To study the contribution of volatile sulfur compounds, the GC-SCD method was applied to 68 commercial wines that had reductive sensory evaluations. The analytes implicated as contributors to reductive characters were hydrogen sulfide, methanethiol, and dimethyl sulfide, whereas carbon disulfide played an uncertain role. KEYWORDS: Gas chromatography; sulfur chemiluminescence detection; wine aroma; volatile sulfur compounds; headspace analysis; wine storage

INTRODUCTION

Over 700 volatile compounds are known to contribute to wine aroma and flavor (1-5), including compounds containing one or more sulfur atoms. Volatile sulfur compounds can be formed by biological and chemical mechanisms at various stages during wine production and storage and often have low aroma detection thresholds (from ng/L to μg/L range). Although there are many sulfur compounds that can potentially be of significance to wine aroma and flavor, we focused this study on volatile sulfur compounds found after fermentation that are relevant to “reduced” aromas and “off-odors”. Typically, these were the more potent, low molecular weight and low boiling point sulfur compounds, along with related acetates and disulfides. There is a great deal that remains to be understood about these compounds, most notably their impact on negative perceptions of wine aroma and the effects of closure type and storage conditions on their formation and stability. A number of low molecular weight volatile sulfur compounds are known to impart unpleasant “reduced”, “onion”, “asparagus”, “burnt rubber”, or “garlic” aromas to wine (2, 6-9). Distinctive “off-odors” can be attributed to specific compounds, such as “rotten egg” from hydrogen sulfide (H2S) (2, 7), “putrid”, “garlic”, or “onion” from methanethiol (MeSH) (7, 10), and *Corresponding author (phone þ61 8 8313 6600; fax þ61 8 8313 6601; e-mail [email protected]).

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Published on Web 08/16/2010

“(canned) corn” or “(cooked) asparagus” from dimethyl sulfide (DMS) at higher concentrations (6, 7, 11). Despite these negative associations, at lower levels DMS can give a pleasant “black currant” aroma and has been shown to enhance fruity notes in the presence of other volatile wine components (12-14). Other volatile sulfur compounds, such as carbon disulfide (CS2), which may be thought of as negative contributors to wine aroma, are not necessarily so at lower concentrations (12). Even H2S may contribute to the bouquet of a young wine and add complexity to wine aroma at low levels at which it is not perceived as a fault (15,16). Table 1 shows aroma descriptors and detection thresholds for 10 volatile sulfur compounds previously found in wine that are generally regarded as causing problems or faults, especially when present at high concentrations. H2S, the most volatile sulfur compound being considered, is used by yeasts to make sulfur-containing amino acids and small peptides (e.g., cysteine, cystine, methionine, glutathione) that are important in yeast cell metabolism and growth (19). It is well established that yeast strain can be a major factor in H2S accumulation during fermentation (19-21). As such, a large amount of H2S can be produced during grape must fermentation, along with other organic sulfur compounds (22). Elemental sulfur, sulfate, or sulfite can be reduced by yeast to produce H2S, with its production also affected by factors such as juice clarity and must nutrients, particularly yeast assimilable nitrogen (YAN) (15, 23-26). Formation of H2S may also occur due to the

© 2010 American Chemical Society

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Table 1. Aroma Descriptors and Detection Thresholds for 10 Volatile Sulfur Compounds Previously Found in Wine odor descriptora

threshold (μg/L)

rotten egg, sewage-like, vegetal rotten cabbage, burnt rubber, putrefaction onion, rubbery, burnt match, sulfidy, earthy sulfurous, cheesy, egg sulfurous, garlic, onion black currant,d cooked cabbage, canned corn, asparagus garlic, rubbery sweet, ethereal, slight green,d rubber, sulfidy vegetal, cabbage, intense onion-like onion

1.1 -1.6 (16)b 1.8 -3.1 (17) 1.1 (6) 50c(18) 10c(18) 25 (6) 0.9 (6) >38 (12) 29 (6) 4.3 (6)

compound hydrogen sulfide methanethiol ethanethiol methyl thioacetate ethyl thioacetate dimethyl sulfide diethyl sulfide carbon disulfide dimethyl disulfide diethyl disulfide a

H2S MeSH EtSH MeSAc EtSAc DMS DES CS2 DMDS DEDS

In-house and ref 8. b Literature source. c In beer. d At low levels.

presence of metals and metal cations (iron, zinc, manganese, etc.) (24, 27). Production of excess H2S can potentially lead to the formation of other sulfur-containing compounds, such as MeSH and ethanethiol (EtSH) and their acetates (15, 25). Although some undesirable sulfur compounds may be removed from wine through copper fining, this approach is not effective for sulfides, disulfides, and thioacetates and is limited to H2S and thiol removal (15, 25). An unwanted side effect of copper fining may be the formation of disulfides and trisulfides exhibiting additional undesirable aromas (25, 28), as well as problems associated with wine instability (29) and additional wine-processing logistics. Fermentation aside, debate remains in the wine industry regarding the propensity of volatile sulfur compounds to form under closures of differing oxygen transfer rates. It could be inferred that, from a finite pool of sulfur compounds at bottling, equilibria may form between thiols, disulfides, and thioacetate esters and other wine matrix components, resulting in the release of potent sulfur compounds during storage under low-oxygen permeable closures (30-33). In addition to the complexity of wine thiol chemistry, other changes to volatile sulfur compounds may be evident. During model studies of wine aging, the concentration of DMS has been shown to increase to potentially unpleasant levels depending on its release from precursor compounds (34) and availability of YAN during fermentation (35). The delayed effect on DMS accumulation and the rerelease of thiols demonstrate that fermentation and processing effects can still manifest themselves some time after bottling, when closure type also plays a critical role. Clearly, the ability to monitor volatile sulfur compounds easily and in a timely manner is therefore of great utility in undertaking such fermentation and storage studies. Due to the volatile nature, reactivity, and relatively low abundance of these sulfur compounds in fermented beverages, specialized techniques are required for their determination. Volatile sulfur compounds have been quantified in wine using solid-phase microextraction (SPME) followed by gas chromatographypulsed flame photometric detection (GC-PFPD) (36, 37), GCflame photometric detection (GC-FPD) (38,39), GC-mass spectrometry (GC-MS) (13, 40), and GC-atomic emission detection (GC-AED) (41). Unfortunately, sampling of such compounds by SPME may suffer from matrix effects (37, 39), and artifact formation or sample losses upon injection (38, 42, 43) can give potentially spurious results. Furthermore, H2S in particular may be less reliably determined using SPME methods (36, 37) and is often missing from analyses employing this technique. In contrast to wine sampling by SPME, other methods involved static headspace or purge and trap techniques along with GC-FPD (7,44,45), GC-AED (35, 46, 47), and GC-sulfur chemiluminescence detection (GC-SCD) (48). Despite the existence of methods for the analysis of volatile sulfur compounds in wine, there is still a need for a rapid, accurate, and reliable method involving minimal

sample preparation so large numbers of samples can be analyzed relatively quickly. In this paper we describe the development and validation of a novel method for volatile sulfur compound analysis using static headspace-cool-on-column (HS-COC) GC-SCD, with ethylmethyl sulfide (EMS) and propyl thioacetate (PrSAc) as internal standards. The method enables the quantitation of 10 volatile sulfur compounds in wine, with its utility demonstrated through application to studies relevant to wine fermentation and storage. MATERIALS AND METHODS Materials. Reference standards of ethanethiol (EtSH, 99.7%), dimethyl sulfide (DMS, 99.8%), diethyl sulfide (DES, 99.3%), dimethyl disulfide (DMDS, 99.8%), diethyl disulfide (DEDS, 99.9%), carbon disulfide (CS2, 99.9%), and ethylmethyl sulfide (EMS, 96.0%) were of the highest purity as supplied by Sigma-Aldrich (Castle Hill, NSW, Australia). S-Methyl thioacetate (MeSAc, 98.8%), S-ethyl thioacetate (EtSAc, 99.5%), and propyl thioacetate (PrSAc, 99.7%) were of the highest purity obtainable from Lancaster Synthesis (Jomar Bioscience, Kensington, SA, Australia). The remaining chemicals listed below were of analytical reagent grade quality or better. Sodium hydrosulfide hydrate (NaSH 3 xH2O, 74.0%), sodium thiomethoxide (NaSMe, 101.8%), and potassium hydrogen tartrate (Fluka) were supplied by Sigma-Aldrich and Merck tartaric acid and sodium chloride (NaCl) were obtained from Rowe Scientific (Lonsdale, SA, Australia). BDH acetaldehyde was supplied by Thermo Fisher (Scorseby, VIC, Australia), Ajax Finechem ethylenediaminetetraacetic acid disodium salt (disodium EDTA) was supplied by Rowe Scientific, ethanol (99.5%, Rowe Scientific) was redistilled inhouse prior to use, and water was obtained from a Milli-Q purification system (Millipore, North Ryde, NSW, Australia). Fermentation-related alcohols, acids, and esters were obtained from Sigma-Aldrich. All solvents and analytical standards were verified for purity by GC-MS and GC-AED or GC-SCD prior to use. EtSH, DMS, DES, DMDS, DEDS, CS2, EMS, MeSAc, EtSAc, and PrSAc were stored at -20 °C to prevent degradation. Containers of NaSH 3 xH2O and NaSMe were sparged with nitrogen and stored in a desiccator at room temperature. Wine Samples. Commercial, bottled wines of various vintages (2004-2008) and varieties (40 red wines and 28 white wines) were obtained from Australian wine producers. The wines were thought to have “reductive” characters on the basis of preliminary sensory assessments by winemakers, wine judges, and trained sensory panelists during informal evaluations. Preparation of Standard Solutions. Stock Standard Solutions of Volatile Sulfur Compounds. Individual stock standard solutions of EtSH, DMS, DES, DMDS, DEDS, CS2, EMS, MeSAc, EtSAc, and PrSAc were prepared by injecting 100 μL of neat standard into 50.0 mL of ethanol contained in a 125 mL Sure-Seal bottle (Sigma-Aldrich) that had been crimp-capped and sparged with nitrogen. The density for each reference standard was used to calculate the actual concentration (approximately 2 g/L), and the solutions were stored at -18 °C for up to 24 months except for EtSH, which was stored for only 6 months. Global Standard Solution of Volatile Sulfur Compounds. A global standard solution of known concentration (with DMS, MeSAc, and

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Table 2. Concentrations of Fermentation-Related Volatiles Added to the Model Wine Used for Calibration Purposes compound ethyl esters ethyl acetate ethyl lactate ethyl propanoate ethyl 2-methylpropanoate ethyl butanoate ethyl 2-methylbutanoate ethyl 3-methylbutanoate ethyl hexanoate ethyl octanoate ethyl decanoate ethyl dodecanoate acetates 2-methylpropyl acetate 2-methylbutyl acetate 3-methylbutyl acetate hexyl acetate 2-phenylethyl acetate

mg/L

20

2

2

compound acids acetic acid

mg/L

40

propanoic acid 2-methylpropanoic acid butanoic acid octanoic acid

8

2-methylbutanoic acid 3-methylbutanoic acid hexanoic acid decanoic acid

2

alcohols 2-methylpropanol butanol 2-methylbutanol 3-methylbutanol hexanol 2-phenylethanol

20

EtSAc each at approximately 50 mg/L and CS2, DES, DMDS, and DEDS each at approximately 12.5 mg/L) was prepared by adding, via syringe, aliquots of each stock standard solution of DMS, DES, DMDS, DEDS, CS2, MeSAc, and EtSAc into a capped and nitrogen-sparged 125 mL SureSeal bottle containing 60.0 mL of ethanol. The solution was stored at -18 °C for up to 6 months. Dilute Standard Solution of EtSH. A dilute standard solution containing a known concentration of EtSH (approximately 10 mg/L) was prepared by adding, via syringe, 600 μL of the EtSH stock standard solution into a capped and nitrogen-sparged 125 mL Sure-Seal bottle containing 100.0 mL of ethanol. The solution was stored at -18 °C for up to 3 months. Stock Standard Solution of NaSH (for H2S) and NaSMe (for MeSH). Due to the impracticality of working with gaseous H2S and MeSH, a suitable alternative employed the sodium salts of these analytes, which were dissolved in cold water (4 °C) and used immediately. Individual stock solutions of known concentration (approximately 300 mg/L) were prepared in amber volumetric flasks. The concentrations of NaSH and NaSMe were calculated using the purity reported in their respective certificates of analysis (Sigma-Aldrich). Dilute Standard Solution of NaSH (for H2S) and NaSMe (for MeSH). Individual dilute standard solutions of known concentration containing NaSH or NaSMe (approximately 7.5 mg/L) were prepared in cold water (4 °C) in 200 mL amber volumetric flasks and used immediately. Internal Standard Mix. An internal standard solution containing known concentrations of EMS (approximately 20 mg/L) and PrSAc (approximately 50 mg/L) was prepared in an amber volumetric flask by diluting the respective stock standard solutions with ethanol. The internal standard solution was stored at 4 °C for 3 months. Preparation of Model Wine. Aqueous ethanol (12% v/v) was saturated with potassium hydrogen tartrate, and the pH was adjusted to 3.2 with tartaric acid solution (40% w/v). Fermentation-derived volatiles (ethyl esters, acetates, alcohols, and fatty acids) were added to approximate the concentrations commonly found in wine (Table 2). Sample Preparation. Wine samples were cooled to 4 °C in their original containers prior to opening, and all sample handling was completed in a temperature-controlled room at 4 °C. An aliquot of wine (10 mL) was added to a 20 mL amber glass headspace vial containing 2 g of NaCl and a 3  8 mm magnetic stir bar. Internal standard solution (25 μL) was added to give known final concentrations of EMS (approximately 50 μg/L) and PrSAc (approximately 125 μg/L). Acetaldehyde (4 μL) was added to each white wine sample vial. The vial was tightly sealed with a white PTFE/blue silicone lined screw cap (Grace Davison Discovery Sciences, Baulkham Hills, NSW, Australia). Instrumentation. Gas Chromatography. The samples were analyzed using an Agilent 6890 gas chromatograph (Forest Hill, VIC, Australia)

equipped with a Gerstel multipurpose sampler (MPS 2XL, Lasersan Australasia, Robina, QLD, Australia) and coupled to either an SCD or AED. Instrument control and data analysis were performed with Agilent GC ChemStation software, rev. B.03.01 and Maestro software integrated version 1.3.3.51/3.3. The gas chromatograph was fitted with a 15 m  0.25 mm FactorFour VFWAXms fused silica capillary column, 0.50 μm film thickness (Varian, Mulgrave, VIC, Australia) connected with a fused silica universal straight connector (Grace Davison Discovery Sciences) to a 60 m  0.25 mm VICI ValcoBond VB-5 fused silica capillary column, 0.50 μm film thickness (Chromalytic Technology, Boronia, VIC, Australia), with a 2 m  0.53 mm retention gap. Helium (Air Liquide ultrahigh purity), linear velocity = 37 cm/s, flow rate = 2.7 mL/min in constant flow mode, was used as the carrier gas. The initial oven temperature was held at 5 °C for 5 min, increased to 150 at 5 °C/min, and held at this temperature for 5 min. The cool-on-column (COC) inlet (Agilent G3440A) (pressurized to 252.69 kPa) was held at 30 °C for 10 min and ramped at the same rate as the oven. The oven and COC inlet were cryogenically cooled with liquid nitrogen. Sulfur Chemiluminescence Detection. An Agilent 355 SCD sulfur chemiluminescence detector coupled to the GC was used with the default SCD parameters recommended by Agilent and sulfur trap gas purifiers on all gas lines (Agilent). The detector base temperature was held at 200 °C and the Dual Plasma Controller at 800 °C. The reagent gases were air (Air Liquide instrument grade), 60.0 sccm; hydrogen (Air Liquide ultrahigh purity), 45.0 sccm; and ozone, generated in situ from air at 41.37 kPa. Atomic Emission Detection. An Agilent G2350A atomic emission detector coupled to the GC was used with AED parameters optimized for sulfur sensitivity. The AED cavity block and the transfer line were held at 250 °C. Helium (Air Liquid ultrahigh purity with SAES PS2GC50 heated getter) was used for the microwave-induced plasma at a flow rate of 25 mL/min, measured at the cavity vent. Oxygen, 379.21 kPa (Air Liquide ultrahigh purity), and hydrogen, 68.95 kPa (Air Liquide ultrahigh purity), were used as the reagent gases. Sulfur (181 nm) and carbon (193 nm) emission lines were monitored. The discharge tube was cooled with water at 65 °C, and the spectrometer was constantly purged with nitrogen at 400 mL/min. Peak Identification. Analytes were identified by comparison of their retention times with those of the corresponding pure reference compounds. Due to the detectors employed, all peaks necessarily arose from sulfur-containing compounds, and analyte identification was unambiguous. Headspace (HS) Equipment and Conditions. The refrigerated sample vials were placed into a Gerstel peltier cooled sample tray (Lasersan) at 4 °C. The vial and its contents were heated to 45 °C for 30 min with stirring at 400 rpm. A Gerstel 1.0 mL HS syringe (Lasersan) was fitted with a custom-made dual gauge cone-tip needle (0.47 mm/0.63 mm, SGE, Ringwood, VIC, Australia), and the syringe heating block was held at 60 °C. A 100 μL static HS sample was injected into the COC inlet at 10 μL/s. The syringe was purged to atmosphere with nitrogen at 10.34 kPa (BOC grade 3.5) for 3 min after injection. Validation. Method precision and calibration linearity were validated by a series of standard addition experiments to model, white, and red wine matrices. Method linearity was determined for nine calibration levels, in duplicate, over the concentration ranges of 0.2-100 μg/L for H2S, MeSH, EtSH, CS2, DES, DMDS, and DEDS, and 1.0-400 μg/L for DMS, MeSAc, and EtSAc. Method precision was determined in all matrices using seven replicate samples spiked at low and high concentrations (5 and 20 μg/L for all analytes except DMS, MeSAc, and EtSAc, which were 50 and 200 μg/L). For quantifying the analytes in batches of unknown samples, duplicate standards (0 and 50 μg/L for all analytes except DMS, MeSAc, and EtSAc, which were 0 and 200 μg/L) were prepared using model wine and analyzed with every set of samples. To check the accuracy of the analysis, duplicate control samples, spiked with 10 μg/L for all analytes except DMS, MeSAc, and EtSAc, which were spiked at 50 μg/L, were included with every set of samples to be quantified, along with blanks. Control samples were prepared in red or white wine with known low levels of the analytes. For red and white wine matrices, 10 mg of disodium EDTA was added to all standard addition, precision sample, and control sample vials. Statistical Analysis. The results reported for validation of the method were derived from the average of duplicate measurements for each

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Figure 1. Typical GC-SCD chromatograms of (A) a white wine and (B) reference standards in a model wine. Analyte abbreviations appear in Table 1, and IS refers to internal standards ethylmethyl sulfide (EMS) and propyl thioacetate (PrSAc). concentration of the analyte (seven replicates for repeatability samples). The limit of detection (LOD) was determined by establishing the minimum level at which the analytes could be reliably detected from the analysis of samples with known analyte concentrations. The limit of quantitation (LOQ) was determined by establishing the minimum level at which the analytes could be quantified with acceptable accuracy from the analysis of samples with known analyte concentrations. RESULTS AND DISCUSSION

Method Development. The initial instrumentation setup consisted of HS-COC-GC-AED that selectively monitored sulfur (181 nm) and carbon (193 nm) (47). The COC inlet and cryogenically cooled GC oven were chosen to overcome the challenges faced in the analysis of inherently reactive and thermally labile sulfur compounds. The analytical method for the determination of fermentative sulfur compounds was then optimized over a period of time as new technologies and instrumentation were acquired. This continual improvement has increased sample throughput to cope with demand because of the widespread application of this analysis to many facets of wine research, particularly winemaking and wine shelf life studies. Method validation was completed using the SCD after incorporation of all optimized parameters. Optimization of Headspace Sampling. Static HS injection into a COC inlet was chosen because it overcomes some of the limitations of SPME techniques and hot injector temperatures. A widebore retention gap, long enough to easily accommodate the injected HS volume, was used, and care was taken to optimize the speed of injection. If the injection rate is too fast, then analytes will be lost backward through the septum purge vent. Therefore, the HS must be injected at a speed slower than the column flow (i.e., 10 μg/L of H2S. As a result, the aroma detection threshold of H2S in wine was revisited and found to be 1.1 and 1.6 μg/L in red and white wine, respectively (16). This means in most of the 68 wines assessed as “reduced”, H2S could be contributing to the reductive aromas, although more investigation is required. To highlight this point, H2S was dismissed as the cause of an “off-odor” by Rauhut et al. for wines presenting “sulfurous off-flavor” (48), and Fang and Qian analyzed commercial wines with no apparent “off-flavor” (36), although H2S was found in both studies at concentrations similar to those in our work. Additionally, Lopez et al. analyzed commercial wines with up to 30 μg/L of H2S yet none had discernible “off-flavors” (37). Methanethiol. MeSH was detected in all but 1 white wine and in 30 of the 40 red wine samples. The highest level of MeSH was 8.0 μg/L in a Chardonnay wine, whereas the highest level in the red wines was 5.0 μg/L in a Shiraz wine (Table 4). MeSH was attributed to “off-odor” in wines with levels >4 μg/L in one study (7), whereas almost 5 μg/L in a wine from another study posed no sulfur-related issues (36). Levels of MeSH around 1.5 μg/L have been associated with “off-odor” in wines (48), but it was apparent that no wine aroma detection threshold was

Table 4. Concentration Ranges of Volatile Sulfur Compounds in Commercial Australian Wines from 2004 to 2008 with Noted “Reductive” Characters (Analyzed May 2009) concentration (μg/L) variety

no. of wines

H2S

MeSH

EtSH

DMS

CS2

DES

MeSAc

DMDS

EtSAc

DEDS

Chardonnay Pinot gris Riesling Sauvignon blanc Sauvignon blanc/Semillon Semillon Verdelho Viognier Cabernet Merlot Cabernet Sauvignon Durif Grenache/Shiraz/Merlot Merlot red wine blend Sangiovese Shiraz Shiraz/Cabernet Sauvignon Shiraz/Viognier

4 1 10 6 4 1 1 1 2 5 1 1 3 1 1 22 2 2

1.5-5.0 2.0 0.5-35.0 0.8-4.0 2.0-13.0 2.0 1.0 0.5 0.5-0.8 nd-1.6 2.0 0.7 0.5-1.2 1.0 nd nd-8.7 0.5-1.0 nd-1.0

3.0-8.0 3.0 nd-3.0 1.7-6.0 1.0-4.0 3.0 1.6 3.0 0.4-1.0 nd-1.5 2.0 0.7 nd-1.6 0.2 nd nd-5.0 1.0-1.2 1.0

nda -0.5 nd nd nd nd-1.0 nd nd nd nd nd nd nd nd nd nd nd-0.7 nd nd

20.0-185.0 11.0 11.0 -37.1 25.0-118.2 25.0-76.0 13.5 47.7 78.0 102.5-106.0 88.0-379.5 61.0 111.0 48.0-235.0 195.0 68.0 28.0-765.0b 85.0-228.4 57.0-112.0

0.5-5.0 0.5 nd-21.1 1.0-13.5 0.5-14.8 2.0 18.6 6.0 3.5-15.6 3.0-20.0 1.0 18.0 8.0-17.0 14.5 4.0 2.0-45.1 4.0-17.4 2.0-6.0

nd nd nd-0.4 nd-0.4 nd-0.4 nd 0.4 nd nd-0.4 nd-0.4 nd 0.4 nd-0.4 0.4 nd nd-0.5 nd-0.4 nd

nd-7.0 nd nd nd nd-2.1 nd nd nd nd nd-10.0 18.0 nd 3.0-8.0 4.7 nd nd-12.5 4.1 -7.5 nd-6.0

nd nd nd nd nd nd nd nd nd-1.5 nd nd nd nd nd nd nd-1.5 nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

a

Not detected. b Extrapolated value, outside the calibration range.

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Figure 3. Concentration of DMS in 68 commercial wines (2004-2008) analyzed in May 2009 by GC-SCD.

available in the literature. Given that MeSH was measured in 57 of the 68 wines shown in Table 4, quite often at levels 3-4 times above which it might pose a problem, it was prudent to accurately determine the aroma detection threshold in wine. We have recently shown that the aroma detection thresholds for MeSH in white and red wines were 3.1 and 1.8 μg/L, respectively (17). This threshold study highlighted the matrix-dependent nature of volatile sulfur compounds, as suggested previously (36), and indicated that MeSH could have contributed to the “reductive” characters of quite a number of red and white wines listed in Table 4. Ethanethiol. EtSH was detected in only one red wine and two white wines (e1.0 μg/L, Table 4), with all being below the reported white wine aroma threshold of 1.1 μg/L (6). In contrast, Park et al. regularly detected EtSH in a set of California wines considered to have “sulfide off-odors” (7), whereas Rauhut et al. barely detected EtSH in a number of wines from various countries displaying “off-flavor”, except for one which had 5.5 μg/L (48). In other studies involving commercial wines without sulfur “off-odor”, EtSH was detected on average 2-3 times above its threshold concentration in numerous Spanish wines (37, 44), whereas no EtSH was present in a number of wines from California, Oregon, and Canada (36). It is doubtful that method sensitivity has affected the various results, so in the absence of elevated must sulfur levels during winemaking (15), the impact on sulfide “off-odor” from EtSH appears to be negligible. Dimethyl Sulfide. Of the volatile sulfur compounds examined, the largest overall concentration range was noted for DMS, with 11.0-185.0 μg/L in the white wines and 28.0-765.0 μg/L in the red wines (Table 4). DMS is an interesting sulfur compound that can be beneficial to wine aroma at low levels (perhaps up to 100 μg/L), increasing the perceived fruitiness (13, 14). At high levels it may mask fruity aromas and impart unpleasant “canned corn”, “cooked cabbage”, or “vegetal” type aromas (6, 7, 11). Certainly the levels encountered in some of the wines we analyzed can be expected to negatively affect the aromas of those wines. Higher levels of DMS are often found in older wines because DMS tends to increase in concentration as wine ages (34, 35). Although the wines were not analyzed at bottling, this trend may be evident within the 68 wine samples we analyzed, in accord with Segurel et al. (13). Figure 3 shows that all of the older wines from 2004 and 2005, except one, contained g100 μg/L DMS, whereas all of the younger wines from 2008, except one, contained 100 μg/L of DMS and most wines from 2007 had less than this amount. It can also be seen from Figure 3 that in general as the wines aged, the levels of DMS encountered for a particular vintage diverged to a greater extent, which may relate to the amounts of DMS precursors remaining in the wine (13, 34).

Siebert et al. Carbon Disulfide. CS2 was detected in all samples except two Riesling wines, with concentrations up to 21.1 and 45.1 μg/L for white and red wines, respectively (Table 4). CS2 was first identified in wines by Lepp€anen et al. in amounts up to 10 μg/L (45), although it may be present at